The generation of replacement tissue in cell culture, commonly known as “tissue engineering”, the provision of surrogate cells for cell-based transplantation therapies, and the identification of new drugs using cell based high-throughput methods all represent important examples of innovative biotechnology-based applications. Stem cells in particular represent an important source for implementing such biotechnological applications because stem cells, in contrast to primary cells, can readily serve as a potentially unlimited source for obtaining differentiated, specific cell types. However, the cultivation of undifferentiated stem cells has proven to be very complex and furthermore, particularly sensitive to external influences.
Aseptic bioreactors require cleaning, sterilization and validation of the standard stainless steel or glass bioreactors by the customer. The use of presterilized disposables that need not be cleaned, sterilized or validated by end users provides savings of costs and multiple hours of labor per run. Furthermore, plastics are lightweight, easy to transport, and require less room than stainless steel or glass vessels. Wave Biotech (Bridgewater, N.J.) has developed a range of presterile, disposable bioreactors that do not require cleaning or sterilizing by the end user. The Wave Bioreactor is made of sheets of flexible, gas impermeable material. The bag is partially filled with media and then inflated with air that continually passes through the bag's headspace. The media is mixed and aerated by rocking the bags up to 40 times a minute to increase the air-liquid interface. However, since a solid housing does not support the bags, the bags become unwieldy and difficult to handle as they increase in size. Furthermore, the wave action within the rocking bag due to the air in the headspace creates damaging turbulent forces. Certain cell cultures, particularly human cell cultures, thrive better under more gentle conditions.
Stem cells may be used for regeneration of heart muscle tissue after a heart infarction and also in vascular regeneration in chronic heart diseases. Furthermore the formation of bone and cartilage from stem cells is well known. Therapeutic applications of stem cells are most beneficial if the stem cells are autologous, i.e. from the person him/herself. The advantages of using autologous cells are evident, cells will always match and rejection of the cells will not happen, so strong immunosuppressive drugs are not necessary as with allografts. In addition, infection risks with hepatitis and HIV are reduced and allergic reactions are diminished. However also allogenous cells may be used, e.g. when no healthy stem cells can be extracted from the patient.
Some cells are adherent, such as mesenchymal stem cells (MSC) and grow only on a surface. These adherent cells can be grown in tissue culture flasks where the cells attach to the plastic surface. It has been shown that they may also be grown on microcarriers (Schop et al, J Tissue Eng Regen Med 2008; vol 2 pages 126-135). WO2009/139703 discloses a method for cell expansion, such as MSC on microcarriers in a plastic bag reactor. The method however uses purified patient tissue, wherein the cells of interest are isolated. The isolated cells are pre-cultured in a T-flask to achieve enough MSC before they are applied to the microcarriers in the plastic bag reactor. This preculture step entails several drawbacks. Additional material is needed, i.e. the T-flask, giving more costs. Furthermore, the cells are first isolated and cultured in the T-flask, meaning additional steps and thus more involvement of highly trained, and thus expensive, persons are needed. In addition, the pre-seeded cells need to be collected and transferred to the plastic bag, which increases the risk of contamination due to the transfer step.
Up to now there is no cell expansion method available that is capable of using crude patient cell samples without needing to purify, isolate and pre-culture the cells before using a disposable bioreactor to expand the cells.
It is therefore an object of the present invention to have a cell expansion method that is capable of using cell and tissue samples that contains more than the cells of interest. The present invention has the advantage that cells of interest need not be isolated from biopsies of patients. The biopsies that still have the cellular composition of the tissue in the patients may be used without laborious isolation and purification steps.
The present invention provides a solution by using microcarriers in combination with a small pore filter and a specific rocking regime of the bioreactor.